Heart disease remains a major cause of morbidity and mortality in the U.S. older population. Heart rate declines and the incidence arrhythmias increase with age. Yet, the molecular basis for heart rate control remains unclear. The hyperpolarization activated current I(f) and T-type calcium current I(Ca-T) are two important contributors to the diastolic depolarization which sets the rate and rhythm of the cardiac pacemaking. In zebrafish (a ne model of cardiac development) mutations which result in slow basal heart rate have been shown to have altered I(f). The neural control of heart rate is mediated by G-protein coupled receptors. Sympathetic (via beta-adrenergic receptors signaling through Gs) and parasympathetic (via m2-muscarinic receptors that signal through pertussis toxin sensitive Gi/Go) produce opposing effects on cardiac function. We have produced knockout cell lines and animals of the three Gi/Go alpha subunits expressed in heart alpha(i2) alpha(i3) and alpha(0). We propose to use these knockouts to understand the signal transduction pathways and the molecular basis of the control of these two ion channels and to use that knowledge to understand the effects in the aging heart. Combining the experience that I have recently obtained in electrophysiology and biophysics with the additional experience in molecular biology that I would gain during this award period, I will be in an unique position to study the mechanism of G-protein regulation of pacemaking activity from cellular electrophysiological events to their functional consequences. The specific aims for this proposal are: Aim 1: To determine the ionic basis for adrenergic and cholinergic control of diastolic depolarization in pacemaker activity. Aim 2: To determine which Gi/Go heterotrimeric protein mediates the cholinergic regulation of I(f) and I(Ca-T), and whether the regulation is direct patch limited or through an indirect pathway. Aim 3: To determine the signal transduction pathway mediating control of ion channels: 3A. To determine whether nitric oxide mediates adrenergic and cholinergic regulation of ion channels and heart rate. 3B. To determine if the G- protein signal is mediated by beta-gamma or alpha subunits. Aim 4: To determine the effect of the alpha subunit gene inactivations on basal heart rate with aging and correlate these changes with changes in G- protein levels using WT and knockout mice. Results from these experiments will add to our knowledge on the normal signal transduction pathways and the impact of altered G protein expression on heart rate regulation with aging.